Difference between revisions of "Part:BBa K2033001:Design"

(→‎Design Notes)
Line 11: Line 11:
 
===Stage 1-Insert Regulator ORFs===
 
===Stage 1-Insert Regulator ORFs===
 
====Phusion PCR====  
 
====Phusion PCR====  
−
The AubR part was first synthesized by Ryan Muller in Spring of 2015. Stage 1 consisted of the insertion of the open reading frame into a cloning vector found [https://benchling.com/hayneslab/f/rvhDwTUE-older/seq-nAjbajRk-modular_receiver_annotated/edit here].
+
The AubR part was first synthesized by Ryan Muller in Spring of 2015. Stage 1 consisted of the insertion of the open reading frame into a cloning vector found [https://benchling.com/hayneslab/f/rvhDwTUE-older/seq-nAjbajRk-modular_receiver_annotated/edit here].  
 +
 
 +
{| border=1
 +
  |+ Caption
 +
|-
 +
  ! Reagent
 +
  ! Volume(uL)
 +
  ! Mix(x5)
 +
|-
 +
  | DNA Plasmid
 +
  | 1.0
 +
  | 5.0
 +
|-
 +
  | 10uM F Primer
 +
  | 1.0
 +
  | 5.0
 +
|-
 +
  | 10uM R Primer
 +
  | 1.0
 +
  | 5.0
 +
|-
 +
  | 10mM dNTPs
 +
  | 1.0
 +
  | 5.0
 +
|-
 +
  | Phusion Pol.
 +
  | 0.5
 +
  | 2.5
 +
|-
 +
  | 5x HF buffer
 +
  | 10
 +
  | 50
 +
|-
 +
  | dH2O
 +
  | 36
 +
  | 180
 +
|-
 +
  | Total
 +
  | 50.5
 +
  |
 +
|}
 +
 
 +
===Stage 2-Insert Promoters===
 +
 
 
===Source===
 
===Source===
  

Revision as of 19:36, 13 September 2016


C(12)-HSL Receiver Device - AubR


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 395
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 395
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 395
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 395
    Illegal AgeI site found at 205
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 184


Design Notes

Here is the inducible promoter sequence:

Stage 1-Insert Regulator ORFs

Phusion PCR

The AubR part was first synthesized by Ryan Muller in Spring of 2015. Stage 1 consisted of the insertion of the open reading frame into a cloning vector found here.

Caption
Reagent Volume(uL) Mix(x5)
DNA Plasmid 1.0 5.0
10uM F Primer 1.0 5.0
10uM R Primer 1.0 5.0
10mM dNTPs 1.0 5.0
Phusion Pol. 0.5 2.5
5x HF buffer 10 50
dH2O 36 180
Total 50.5

Stage 2-Insert Promoters

Source

This HSL receiver was recovered from a metagenomic project on soil. The specific source is unknown.

References

(1) Nasuno, E., N. Kimura, M. J. Fujita, C. H. Nakatsu, Y. Kamagata, and S. Hanada. "Phylogenetically Novel LuxI/LuxR-Type Quorum Sensing Systems Isolated Using a Metagenomic Approach." Applied and Environmental Microbiology 78.22 (2012): 8067-074. Web